The tripartite interaction between dietary fiber and protein intake, microbial fermentation and host cell metabolism, is on display in the colon where epithelial cells are routinely exposed to high concentrations of the respiratory poison, hydrogen sulfide (H2S). H2S is also beneficent; it is used for signaling via persulfidation, a posttranslational modification, and is a substrate for the electron transfer chain, generating ATP. Thiosulfate is the primary product of H2S oxidation in colon and its synthesis involves three mitochondrial enzymes: sulfide quinone oxidoreductase (SQR), a persulfide dioxygenase (PDO), and a sulfurtransferase. We have found that the apical localization of all three enzymes in colonic crypts is influenced by the presence or absence of microbiota and that these enzymes are significantly overexpressed in colon cancer. We now seek to expand from our initial focus on the enzymology of human sulfide oxidation enzymes to the interconnection between the oxidation pathways for H2S and butyrate, which is the preferred fuel for colonocytes and is furnished by microbial metabolism. We posit that prioritization of H2S over butyrate oxidation (to avert poisoning), is achieved via coenzyme A persulfide (CoASSH) produced by SQR and oxidized by PDO. CoASSH inhibits butyryl-CoA dehydrogenase (ACADS), explaining why native ACADS has long been purified as a ?green? enzyme due to an inhibitory charge transfer complex between CoASSH and the flavin cofactor. To test this hypothesis, we will determine the kinetics of CoASSH synthesis and its inhibition of ACADS in vitro and of butyrate oxidation in cells with normal or ablated SQR or PDO expression. We will also determine the structures of human SQR and PDO (with a tightly bound inhibitor) to inform our kinetic and cellular studies and will assess the competition between SQR and complexes I and II for a limited coenzyme Q pool. Using a proteomic method, we have identified >1800 persulfide targets and we will investigate the role of H2S signaling in modulating metabolism in nonmalignant and malignant colon epithelial cells. For this, we will use radiolabel tracing and metabolomics analyses to assess the influence of sulfide on central carbon, nucleotide and lipid metabolism, evaluate the role of a sulfurtransferase in catalytic persulfidation of protein targets, and assess the influence of gut microbiota and diet on host sulfide metabolism. The impact of our broad and rigorous attack on the challenging study of sulfide-mediated signaling and metabolic modulation, supported by strong ongoing collaborations (with a physician scientist, cancer cell biologists, a microbiologist and physical chemists), will be to provide fundamentally important biological insights at the relatively unexplored host-microbiome interface.